Deletion of TAL does not decrease GNG flux.

A—The novel pathway as suggested by Hannaert [25]: Erythose 4-phosphate (E4P) is condensated with dihydroxyacetone phosphate (DHAP) by fructose-1,6-bisphosphate aldolase into S1,7bP, which is dephosphorylated by SBPase into S7P. That is used as a substrate by transaldolase, together with glyceraldehyde 3-phosphate (GA3P), and converted into F6P and E4P, which can enter another cycle. B–PCR detection of a 500-bp product from the TAL ORF, proving deletion of the gene (top panel). PCR with oligos annealing to to the UTRs of the TAL gene, amplifying the TAL gene in the parental cell line (1,402 bp), replaced by phleomycin resistance gene (775 bp) in Δfbp.sbp.tal, and both phleomycin resistance and puromycin N-acetyltransferase (1,006 bp) in Δtal (bottom panel). C–Growth curves of the parental (2T1^T7-Cas9), Δtal, and Δfbp.sbp.tal cell lines in HMI-11 medium supplemented with glucose, or glycerol only (and 10% FBS). D—LC-MS metabolomics in CMM supplemented with 5 mM ¹³C₃-glycerol. G6P –glucose 6-phosphate, F1,6bP—fructose 1,6-bisphosphate, S7P - sedoheptulose 7-phosphate. 2T1 –parental cell line in medium with glucose, 2T1 ¹³C –parental cell line in medium with ¹³C-glycerol, Δtal¹³C –Δtal cell line in medium with ¹³C-glycerol, Δfbp.sbp.tal—Δfbp.sbp.tal cell line in medium with glucose, Δfbp.sbp.tal¹³C - Δfbp.sbp.tal cell line in medium with ¹³C-glycerol.